PROJECT SUMMARY Hematopoietic stem cells (HSC) have well established clinical applications in the treatment of heritable and acquired blood disorders. However, their therapeutic potential could be significantly broadened by engineering novel methods to generate HSC de novo from pluripotent stem cells or from directly reprogrammed adult cells. Toward this goal, we have established endothelial cell (EC) niche based culture methods that provide the necessary conditions to support the specification and self-renewal of HSC from embryonic hemogenic precursors, and more recently, from adult ECs using transcription factor (TF)-mediated conversion that bypasses a pluripotent intermediate. We hypothesize that recreating the signals necessary and sufficient to develop a clinically meaningful system for HSC generation in vitro will necessitate a comprehensive, systems approach to deconstruct the niche provided signals required for HSC specification and self-renewal. Thus, the overall goal of this grant is to leverage unique expertise of the collaborating laboratories to elucidate the signaling interactions regulating HSC specification and self-renewal from embryonic hemogenic precursors or TF-reprogrammed adult EC in the context of the EC niche. Our approach consists of three overlapping aims. The first aim will identify EC niche-provided signals necessary for embryonic HSC specification and self-renewal. The second aim will identify the unique HSC programs induced by these signals that regulate the transition from embryonic hemogenic precursor to bone fide repopulating HSC. The third will identify comparable programs that regulate the transition from adult EC to HSC during TF-mediated reprogramming in the EC niche. Key to these studies will be innovative functional assays, transcriptional profiling methods, and computational approaches that will enable us to resolve cellular complexity of niche cells and their interactions with developing embryonic or reprogrammed HSC at the single cell level. The role of identified signal factors in stage-specific support of HSC specification will be validated and further refined in vitro by gain and loss of function studies in the context of niche EC. Furthermore, to extend these studies to stromal cell-free systems as a step toward clinical translation, we will also test the contribution of identified signal factors in HSC specification and self-renewal in the context of stage-specific modulation of Notch activation using engineered Notch agonists. To achieve the goals of this proposal, we have developed a multidisciplinary collaboration involving unique expertise in each of our laboratories, including basic HSC and EC niche cell biology, direct TF based cellular conversion, clinical HSC transplantation, genome wide assessment of rare stem cell populations at single cell resolution, and innovative computational approaches to deconstruct core signal pathways regulating developmental transitions. Altogether, we expect the proposed studies will ultimately guide the design of novel strategies for deriving and expanding HSC in vitro for therapeutic applications.